Profilin is a small actin-binding protein and is expressed at high levels in mature pollen where it is thought to regulate actin filament dynamics upon pollen germination and tube growth. The majority of identified plant profilins contain a MAP kinase phosphorylation motif, P-X-T-P, and a MAP kinase interaction motif (KIM). In in vitro kinase assays, the tobacco MAP kinases p45(Ntf4) and SIPK, when activated by the tobacco MAP kinase kinase NtMEK2, can phosphorylate the tobacco profilin NtProf2. Mutagenesis of the threonine residue in this motif identified it as the site of MAP kinase phosphorylation. Fractionation of tobacco pollen extracts showed that p45(Ntf4) is found exclusively in the high-speed pellet fraction while SIPK and profilin are predominantly cytosolic. These data identify one of the first substrates to be directly phosphorylated by MAP kinases in plants.